1. Field of the Invention
The present invention generally relates to the cryologic preservation of cellular tissue. More particularly, the present invention relates to the cryologic preservation of epidermis sheets obtained by cellular culture.
2. Brief Description of the Background Art
Despite recent progress in treating burnt tissue (i.e., tissue which is characterized by sufficient amounts of denatured protein to result in cell injury or death), the death-rate of burn victims still remains unacceptably high. Such fatality levels are, of course, highly dependent on the patient's age and on the percentage of burnt body area. The death-rates are, in fact, dramatically high for third degree burns (i.e., when full-thickness dermis has been destroyed) of 50-60% or more body area. In these cases, especially, in addition to the immediate problems of advanced cardio-vascular shock and the threats of somewhat less-imminent sepsis or hydro-electrolytic impairment, yet another (slightly delayed) problem is caused by the absence of a cutaneous covering over a large body area. The difficulty with such missing coverings, or open areas, is that they tend to not spontaneously recover, since they have been completely damaged or destroyed throughout their whole thickness.
When the normally closed epithelial barrier is opened to the outside environment, a chronic pathological condition is created which, together with the above identified infective and metabolic complications, progressively worsen the patient's local and systemic conditions. The sum total of these problems often overwhelms the burn victim, who freguently dies.
It is therefore evident, once the emergency phase of treatment has been overcome, that the main medical problem is to cover the cutaneous deficit as quickly and as effectively as possible. According to conventionally accepted medical practice, wound areas are covered by transplanting split thickness tissue pieces removed from healthy unburnt regions (autografts). However, when the burnt area exceeds, for example, 60-70%, there is usually insufficient donor tissue available for grafting. Moreover, the auto graft donor site itself provides yet additional areas which lack whole cutaneous coverage. These drawbacks must be considered together with the facts that autograft donor sites also tend to recover slowly and may yield deleterious healing residues.
It is also medically acceptable to cover the cutaneous deficit by the transplantion of homologous cadaver skin, lyophilized heterologous skin (from, for example, pig), synthetic artificial cutis and the like. However, these alternate materials, being allogenic will slough spontaneously within 1-6 months, and they must eventually be replaced with autologous tissue and are thus, at best, temporary in nature.
Methods recently disclosed in U.S. Pat. Nos. 4,016,036; 4,304,866 and 4,456,657 teach methods for culturing human epithelial films obtained from cutaneous keratinocytes. These films, of course, may be autologous and therefore would allow burnt areas to be covered without being subject to later rejection or sloughing. These methods in these U.S. patents are based on the removal of a small (2-3 cm.sup.2) piece from a healthy donor area. An epithelial cellular suspension obtained using the donor tissue is then cultured and expanded through subsequent inoculations of the primary culture. When the secondary and tertiary cultures become confluent, multi layered sheets of epithelium can be obtained.
Thus, several serious problems are potentially overcome, such as the poor availability of residual healthy skin in the seriously burnt patient or the need to use expensive pig, cadaver, or artificial cutis, each of which may not be readily available and whose use may be strictly regulated because of antibody reaction.
On the other hand, the keratinocytes cultures obtained according to the above methods require from 3-4 weeks until they are suitably expanded so that film sheets may be obtained. In other words, the burnt patient must remain without protection of the burnt areas for the initial 3-4 weeks. This initial period is, of course, the most critical and so, that period during which the graft of healthy cutis would be most highly beneficial. Additionally, the cultured epithelial sheets taught in these U.S. patents must be used within a few hours of harvesting since it has not been possible to develop a conservation method which can maintain expanded epidermis sufficiently to allow their travel to distant user centers.
The availability of frozen cultured epithelium is advantageous also for those patients that require several transplantations, since it facilitates a better coordination between culture and surgical schedules.
Therefore, it is now possible to realize the institution of "tissue banks" or other depositories of allogeneic expanded epidermis which could be relied upon in the event of emergency.
Readily available cultured allografts in a frozen state can have several clinical applications. For example the present inventors have evidence that deep second degree burns heal much faster when grafted into cultured allografts. Limited full thickness lesions clinically appearing as third-degree burns were reepithelialized by the recipient keratinocytes, whose growth and probably migration were strongly stimulated by cultured allografts.
Cultured allografts could also be applied in most of donor sites utilized for split thickness mesh grafts. This results in a nice healing of those areas even after 4 days.
The method described herein can be successfully utilized also for cryologic preservations of mucosa expanded in vitro. In this regard, the present inventors have determined that expanded mucosa can be obtained in vitro starting from a punch biopsy following the methodology described for production of epithelial films from cutaneous keratinocytes. The present inventors have also determined that the in vitro expanded mucosa can be successfully transplanted in patients that require oral mucosa transplant.